This research project aims to develop and apply cutting-edge analytical chemistry technologies and methodologies such as single-molecule biosensors (SMB) and single-molecule chemical microscopy for real-time tracking of a cascade of biological events (e.g., T cell immune response) in real-time at the SM level. The study of thermodynamic (affinity, stoichiometry) and kinetic (mechanism, rate constant) parameters of ligand-receptor binding reactions on the surface of single cells at the SM level is an essential step in gaining an understanding of the onset of biological cascades, disease infection and the design of potential drugs. These innovative approaches will prove value in unraveling mysteries that presently prohibit us from completely understanding diseases such as cancer and AIDS from their onset and development through their diagnosis and treatment. These studies will also provide unique opportunities to understand fundamental theories (e.g., lock-and-key model) at the SM level. Ultimately, this research may lead to the discovery of new cellular signaling pathways and the invention of novel technologies for biomedical research. The invention and development of novel molecule devices (e.g., SMB) for biomedical applications will also raise the new challenges and open up the new opportunities for biomedical engineers. The general scheme is replied upon the novel approaches of state-of-the-art ultrasensitive analysis and single- molecule detection (SMD) recently described and demonstrated in our laboratory. Two interrelated research aims are: 1) To develop novel SMB and SM chemical microscopy for real-time tracking of single-cell immune response. Instrumentation will be constructed using real-time coupling of SMB with SM fluorescence microscopy. This instrumentation will be capable of determining the real-time thermodynamic and kinetic profiles of cellular signaling pathways associated with single ligand-receptor interaction on the surface of single cells, and 2) To demonstrate at the single-cell and SM level, real-time monitoring of the regulation of T cell immune response. A SMB will be used to trigger a single ligand-receptor binding event on the surface of a single T cell. The associated cellular signaling pathways (e.g., hydrolysis of inositol phospholipids in the plasma membrane, increase in intracellular second messenger levels) will be monitored in real-time using SM chemical microscopy.